Signal transduction mediated by IL-4 in hematopoietic cell lines was shown to induce tyrosine phosphorylation of cellular proteins. The most prominent IL-4-induced phosphorylated substrate was a 170 kd protein. designated 4PS (IL-4-induced phosphotyrosine substrate). the dose required to observe tyrosine phosphorylation of 4PS correlated with mitogenicity induced by IL-4. It was shown that IL-4. insulin and IGF-1 share the overlapping signaling pathways in hematopoietic cells by utilizing 4PS. Moreover. 4PS was shown to be related but not identical to IRS-I. the principal substrate of insulin and IGF-1 in cells of connective tissue origin. 32D cells. a factor-dependent murine myeloid cell line. did not proliferate in response to the IL-4 or insulin. even when either receptor was overexpressed. Examination of tyrosine phosphorylation pattern induced by IL-4 or insulin revealed that there was no detectable 4PS expressed in 32D cells. When an IRS-I CDNA was introduced into either parental or receptor overexpressed 32D cells. the mitogenic sensitivity to IL-4 or insulin was reconstituted. These results indicate that growth factors that bind to unrelated receptors can utilize similar signaling elements in hematopoietic cells. and that IRS-I and 4PS are functionally similar proteins that are essential for insulin- and IL-4-induced proliferation. Functional domains of IL-4 receptor importance for signal transduction were studied by introducing a series of human IL-4 receptor mutants into IRS-I-expressing 32D cells. Results suggested that an acidic region within the cytoplasmic domain between amino acids 437 and 557 was critical for IL-4 signaling. This region contains a sequence motif 488- PLXXXXNPXYXSXSD-502. also found in the intracellular domain of insulin and IGF-I receptors. and was designated I4R motif. Point mutation on tyrosine 497 within this I4R motif greatly reduced IRS-I phosphorylation and cell proliferation in response to human IL-4. These results suggest that the central tyrosine within I4R motif is important for IL-4 signaling.